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. 2020 Jun 25;10(7):317. doi: 10.1007/s13205-020-02307-0

Antibacterial metabolites from Bipolaris specifera, an endophytic fungus from the endemic medicinal plant, Zingiber nimmonii (J. Graham) Dalzell

Madhuchhanda Das 1, Harishchandra Sripathy Prakash 2, Monnanda Somaiah Nalini 1,✉
PMCID: PMC7316947  PMID: 32626638

Abstract

Eleven fungal endophytes were isolated from the plant parts of Z. nimmonii (J. Graham) Dalzell, an endemic species of the Western Ghats, India, a biodiversity hotspot area. The endophytic isolates were characterized by the sequencing of the Internal Transcribed Spacer (ITS) regions and designated as strains by depositing ITS sequences in the Gen Bank sequence database. All the strains were cultured in Potato Dextrose broth (PDB, 500 mL) contained in Erlenmeyer flasks to obtain the secondary metabolites. The culture filtrate was extracted with ethyl-acetate (EA) three times and concentrated by flash evaporation to obtain EA crude dry extract. The strains were evaluated for the antibacterial potentials against six pathogenic bacterial strains viz., Bacillus subtilis (MTCC 121), Staphylococcus aureus (MTCC 7443) Pseudomonas aeruginosa (MTCC 7093), Escherichia coli (MTCC 729), Enterobacter aerogenes (MTCC 111) and Klebsiella pneumoniae (MTCC 661). Nine endophytic fungal extracts except Alternaria consortiale and Hypocrea lixi showed inhibitory activities against at least two of the six test bacterial strains. Bipolaris specifera (KM114290) exhibited the highest inhibition zones ranging from 15.1 ± 0.3 to 26.7 ± 1.1 mm (diameter), against all six test bacteria in the agar disk diffusion assay, and with Minimum Inhibitory Concentrations (MIC’s) of 0.04–0.14 mg/mL, followed by Aspergillus terreus. B. specifera extract was therefore selected and characterized for the identification of antibacterial compounds by chromatographic techniques. Seven antibacterial compounds viz., (1) Bicyclo[3.2.0]heptan-2-one, 6-hydroxy-5-methyl-6-vinyl; (2) Adipic acid divinyl ester; (3) 1,4-Naphthoquinone, 6-acetyl-2,5-dihydroxy; (4) Decanedioic acid, 3,7-dimethyl ester; (5) (Z)-4-Hexenoic acid 2-acetyl-2-methyl-ethyl ester and (6) Butanoic acid 2-acetyl-3-methyl-methyl ester and (7) Caffeic acid, were identified through liquid and gas chromatography. These compounds are mainly volatile esters of fatty acids, phenolics and adipic acid found rare in nature. This study envisages the possible drug discovery using endophytes from traditional and endemic medicinal species.

Keywords: Endophytic fungi, Zingiber, Western Ghats, Antibacterial, Bipolaris specifera, GC–MS

Introduction

Antibiotics are secondary metabolites that are produced by some microorganisms or plants and restrict other pathogenic microorganisms. The overuse of such antibiotics on a daily basis makes the pathogens resistant to the drug. Medicinal plants are overexploited for the search of newer and potential bioactive compounds, which is a threat to biodiversity. The discovery of novel antimicrobial compounds from endophytes is an important alternative source to overcome these problems (Pimentel et al. 2011). These organisms reside in the intercellular spaces of host plants without causing any overt symptom or damage. Fungi are the most common endophytic organisms isolated and are present in all plant species (Schulz and Boyle 2006). New bioactive natural compounds have been isolated from fungal endophytes (Nalini et al. 2019). The isolated antimicrobial compounds belong to several structural classes like phenols, flavonoids, steroids, alkaloids, peptides, terpenoids and quinones (Yu et al. 2010).

The genus Zingiber consists of 85 aromatic species, and is widely distributed in East Asia and tropical Australia. Z. officinale Roscoe or ‘ginger’ is an ingredient in the traditional Indian medicine for relief from several ailments (Sabulal et al. 2006). Z. nimmonii (J. Graham) Dalzell, is an endemic ginger species of the Western Ghats, a biodiversity hotspot in India. The aromatic oil from the rhizome is rich in Caryophyllene compounds that are reported to possess antimicrobial potentials (Sabulal et al. 2006).

The isolation of fungal endophytes from the plant parts and their antioxidative potentials is reported from our previous study (Das et al. 2017a). In the present study, the evaluation of antibacterial potentials from the fungal endophytes of Z. nimmonii against six test bacterial strains and the characterization of the secondary metabolites, from the potential strain, B. specifera by chromatographic techniques are investigated.

Materials and methods

Plant collection and the isolation of endophytic fungi

Z. nimmonii was collected from the Talacauvery sub cluster (012°17′ to 012°27′ N and 075°26′ to 075°33′E) of Western Ghats, in Kodagu district, Karnataka state, India, during November, 2011. Endophytic fungal isolations were carried out aseptically, according to Tejesvi et al. (2005). The details of the methods are as follows: The plant parts, comprising of roots, rhizome, leafy stem, leaves and the inflorescence were processed for surface sterilization in 70% (v/v) ethanol for 1 min, and immediately with sodium hypochlorite (3.5%) for 3 min and thoroughly washed in sterile distilled water for 3–4 times. Dried plant parts were cut into 1.0 cm × 0.1 cm × 0.1 cm pieces under sterile conditions. A total of 900 plant fragments were plated on Water Agar media (2% WA, w/v) supplemented with the antibiotic streptomycin (50 mg/l) to suppress the bacterial growth and favour the growth of fungal endophytes. The plates were sealed with Clean Wrap™ and incubated at 28 ± 2 ℃ with 12 h of light and dark cycles for 4–6 weeks. The plates were observed periodically. Fungal hyphae and fruiting structures emerging from the plated fragments of tissues were cultured on PDA at 28 ± 2 ℃ for 10–15 days and maintained as pure culture at 4 ℃ for further use.

Molecular characterization of endophytic fungi by DNA sequence analysis of the ITS (internally transcribed spacer) region

Isolation of genomic DNA and amplification

Actively grown mycelial plugs from 11 morphologically different endophytic fungi were inoculated into potato dextrose broth (PDB, Hi Media, Mumbai, India). The isolates were grown in still culture at 28 ± 2 ℃ for 7–10 days. The genomic DNA was extracted from the freeze-dried fungal mats by cetyltrimethyl ammonium bromide (CTAB) method with slight modifications (Ausubel et al. 1994). The DNA concentration was estimated by measuring the absorbance at 260 and 280 nm (Thermo Scientific Nanodrop 2000/2000c, Bangalore, India). Target regions of the rDNA ITS 1 and 2 regions and 5.8 rRNAgene were amplified using primers ITS 1 and ITS 4 (Das et al. 2017a). The amplified product was subjected to sequencing at Chromous Biotech Pvt. Ltd. Bangalore, India. The endophyte sequences were aligned with the reference sequences using the BLAST algorithm and submitted to the NCBI GenBank nucleotide collection.

Fermentation and extraction of metabolites

The pure cultures of 10 days old isolates were inoculated into 500 ml of PDB contained in Erlenmeyer flasks in duplicates and kept for incubation for 3 weeks at 28 ± 2 ℃. The fermentation broth of each endophyte was extracted with ethyl acetate thrice at room temperature and further concentrated by a Rotary flash evaporator (Superfit Model, PBU-6D, India). The residue obtained was designated as the crude dry extract and stored in glass vials, until use.

Detection of antibacterial activity

Test organisms

Two Gram-positive bacteria viz. Bacillus subtilis (MTCC 121) and Staphylococcus aureus (MTCC 7443) and four Gram-negative bacteria viz. Pseudomonas aeruginosa (MTCC 7093), Escherichia coli (MTCC 729), Enterobacter aerogenes (MTCC 111) and Klebsiella pneumoniae (MTCC 661) were used. These test organisms were procured from the Department of Studies in Microbiology, University of Mysore, Karnataka, India.

Antibacterial activity

The inhibitory effect of the endophytic fungal extract was tested by disk diffusion method (Bauer et al. 1966). The crude extract of endophytic fungi was dissolved in dimethyl sulfoxide (DMSO) and tested on Mueller–Hinton agar medium seeded with the test bacterium at 250 µg/disk (5 mm diameter, Whatman no. (1) concentration. Streptomycin (10 µg/disk) was used as positive control and paper disk loaded with only DMSO was negative control. The test plates were incubated for 24 h at 35 ± 2 °C and inhibition zones were measured.

Determination of MIC and minimum bactericidal concentration (MBC)

MIC was determined by modified broth dilution method (Xu et al. 2008), using sterile 96-well microplate (Tarsons, Kolkata, India). The wells were filled with reaction mixture containing 90 µL bacterial suspensions (106CFU/ mL) and 10 µL of test sample with different concentrations (2 mg/mL to 0.02 mg/mL). The culture medium with 1% DMSO was used as the negative control and streptomycin sulphate (0.4 mg/mL to 0.01 mg/mL) was the positive control. The microplates were incubated for 24 h at 35 ± 2 °C. After the incubation, 10 µL of the indicator 3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (0.5 mg/mL phosphate buffer saline) was added to visualize the microbial growth. The lowest sample concentration at which no blue colour appeared was determined as MIC. The wells containing MIC concentration and above was inoculated onto agar medium to check cell viability. The lowest concentration with no viable cells was determined as MBC.

TLC and bio-autography

The ethyl-acetate extract of endophytic fungi with antibacterial activity was subjected to TLC for the separation of active compound. The solvent systems used as liquid phase was a mixture of chloroform: ethyl acetate: formic acid in the ratio of 20:16:4. The developed chromatograms were subjected to agar overlay bio-autography (Rahalison et al. 1991).

Identification of the antibacterial compounds through LC–MS analysis

Analyses of antibacterial compounds by LC–electrospray ionization (ESI)–MS were carried out using Synapt G2 High definition mass spectroscopy (Waters, Milford, MA, USA). LC separation was performed on a reversed-phase Acquity UPLC BEH C18 column (2.1 × 50 mm) with 1.7 µm particle size (dp) (Waters™, Milford, MA, USA). The LC condition was as follows: solvent A (0.1%) formic acid and solvent B (100%) acetonitrile. A gradient elution, 0–2.5 min 95% A, 5% B; 2.5–4.0 min 10% A, 90% B; 4.0–5.0 min 95% A, 5% B was used with flow rate 0.7 ml/min. The UV detector was set to an absorbance wavelength of 280 to 340 nm. The LC elute was introduced directly into the ESI interface without flow splitting. The scan range of ESI–MS was m/z 100–1000. The drying gas (N2) flow was 500 /Lh. The ESI capillary voltage was 3 kV ion mode.

GC–MS analysis of antibacterial compounds

GC–MS (GC-17A with QP5000 MS, Shimadzu Corp., Kyoto, Japan) was used to analyse the volatile and aliphatic compounds. A SPB-1 column (30 m × 0.32 mm with film thickness 0.25 μm; Sigma-Aldrich, St. Louis, MO, USA) was used and sample dissolved in acetone (2 μL) was injected with split ratio of 20:1. The analysis carried out with oven temperature programmed at 50 °C (hold 3 min) and raised to 260 °C at a rate of 5 °C/min. The injection port temperature was 250 °C, transfer temperature was 200 °C and ion source temperature was 180 °C. Helium was used as carrier gas at a flow rate of 1 ml/min. The instrument was calibrated to scan range m/z 40–400. The compounds were identified by computer matching of their mass spectral fragmentation patterns with the NIST-MS library.

Results and discussion

Isolation and the identification of endophytic fungi

900 surface sterilized plant fragments of Z. nimmonii plant parts yielded 471 endophytic fungal isolates based on their colonization frequency (% CF; Das et al. 2017a). The isolates were identified morphologically, microscopically and with the DNA sequence analysis of the ITS region, and assigned to 11 species as Bipolaris specifera, Aspergillus terreus, Alternaria tenuissima, Neocosmospora haematococca (syn. Nectria haematococca), Fusarium chlamydosporum, Sarocladium kiliense, Neocosmospora solani (syn. Fusarium solani), Hypocrea lixi, Fusarium equiseti, Trichoderma harzianum and Alternaria consortiale. The detailed identification of endophytic fungi with their Genbank accession numbers is reported in our previous investigation (Das et al. 2017a). The use of Zingiber in the traditional medicine is well documented. Z. nimmonii, a wild congener of ginger, is an endemic medicinal plant of the Western Ghats-a biodiversity hotspot. The endophytic fungal assemblage of this plant and their antioxidant potentials are reported in our previous study (Das et al. 2017a). Ethnomedicinal species of this region are known to harbor endophytes with bioactive potentials (Das et al. 2017b, 2018).

Antibacterial activity

The antibacterial activity of the isolated strains was tested against six pathogenic bacteria and the resulting inhibition zone and minimal inhibitory concentration (MIC) is presented in Tables 1 and 2, respectively. B. specifera exhibited highest inhibition zone against all the pathogens followed by A. terreus and N. haematococca. The inhibition zone formed by B. specifera is represented in Fig. 1. B. subtilis was resistant against all the extracts except B. specifera. A. consortiale and H. lixi did not show any activity. All the endophytic fungal extracts except that of A. consortiale and H. lixi showed inhibitory activity against E. aerogenes and E. coli between concentrations ranged from 1.8 to 0.04 mg/mL and 2 to 0.04 mg/mL respectively. Previously, there is no documentation of the antibacterial potential of fungal endophytes from this plant. Sabulal et al. (2006) reported the antibacterial activity of Z. nimmonii rhizomatous oil, with the inhibition zone ranged from 7.7 ± 0.6 mm to 11.3 ± 0.6 mm, against B. subtilis, S. aureus, E. coli and K. pneumoniae. In the present study, B. specifera showed strong inhibition properties against these bacteria (inhibition zone ranged from 15.1 ± 0.3 mm to 26.7 ± 1.1 mm). These evidences substantiate the endophytes as alternative sources of plant medicine. The extract of B. specifera showed good antibacterial properties against all the pathogens. Therefore, it was further characterized for the identification of antibacterial compounds by TLC and bio-autography.

Table 1.

Antibacterial activity of fungal endophytes isolated from Z. nimmonii against six pathogenic bacteria

Extracts of endophytic strains Gen bank accession no. Gram-positive Gram-negative
Bacillus subtilis Staphylococcus aureus Pseudomonas aeruginosa Escherichia coli Enterobacter aerogenes Klebsiella pneumoniae
Bipolaris specifera KM114290 17.8 ± 0.7 18.9 ± 1.0 15.1 ± 0.3 26.7 ± 1.1 25.4 ± 0.3 16.0 ± 0.6
Aspergillus terreus KM396303 – 13.9 ± 0.2 7.2 ± 0.1 14.8 ± 0.4 16.5 ± 0.5 7.5 ± 0.2
Alternaria tenuissima KJ547594 – 9.9 ± 0.3 – 13.0 ± 0.3 9.2 ± 0.2 10.2 ± 0.4
Alternaria consortiale KM114288 – – – – – –
Nectria haematococca KM396304 – 12.5 ± 0.5 11.1 ± 0.1 14.2 ± 1.0 17.2 ± 0.5 11.7 ± 0.1
Fusarium chlamydosporum KM396301 – 10.6 ± 0.4 9.2 ± 0.2 11.5 ± 0.5 16.8 ± 0.3 7.0 ± 0.5
Fusarium solani KJ547596 – 8.6 ± 0.4 10.5 ± 0.2 11.2 ± 0.1 7.5 ± 0.2 9.7 ± 0.5
Fusarium equiseti KM396306 – – – 9.3 ± 0.1 8.2 ± 0.1 –
Hypocrea lixi KM396302 – – – – – –
Sarocladium kiliens KM396305 – 8.1 ± 0.1 9.5 ± 0.3 10.2 ± 0.2 8.5 ± 0.1 –
Trichoderma harzianum KJ547595 – – 11.2 ± 0.2 12.7 ± 0.5 11.5 ± 0.1 –
Streptomycina – 32 ± 0.1 31.5 ± 0.5 33 ± 0.3 20 ± 0.1 22 ± 0.1 30 ± 0.2
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Data are reported as mean ± SD (in mm) of three independent analyses (n = 3)

aStreptomycin 10 µg disk used; ‘–’ indicates the absence of inhibition zones in the disk diffusion assay with the endophytic fungal extracts as tested against the test bacterial pathogenic strains

Table 2.

Minimal inhibitory (MIC mg/mL) concentrations and minimum bactericidal concentrations (MBC mg/mL) of fungal extracts

Endophytic fungal strains/extracts Test bacterial strains
S. aureus B. subtilis P. aeruginosa E. coli E. aerogenes K. pneumoniae
MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC
B. specifera 0.1 0.12 0.1 0.12 0.14 0.16 0.04 0.06 0.04 0.06 0.14 0.16
A. terreus 0.16 0.18 – – 1.4 1.6 0.16 0.16 0.12 0.14 1.2 1.4
A. tenuissima 1.0 1.0 – – 1.8 2.0 0.18 0.2 1.0 1.2 0.8 1.0
A. consortiale – – – – – – – – – – – –
N. haematococca 0.6 0.8 – – 0.8 1.0 0.16 0.18 0.10 0.12 0.6 0.8
F. chlamydosporum 0.8 1.0 – – 1.0 1.2 0.6 0.8 0.12 0.14 1.4 1.6
F. solani 1.2 1.4 – – 0.8 1.0 0.8 1.0 1.2 1.4 1.0 1.2
F. equiseti – – – – 1.8 2 1.0 1.2 1.2 1.4 – –
H. lixi – – – – – – – – – – – –
S. kiliens 1.2 1.4 – – 1.0 1.2 0.8 1.0 1.2 1.4 2.0 –
T. harzianum – – – – 0.8 1.0 0.6 0.6 0.6 0.8 2.0 –
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Fig. 1.

Fig. 1

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Inhibition zone formed by fungal endophytic extracts. The fungal strains, B. specifera (BS) and A. terreus (AT) were isolated as endophytes from the surface sterilized root and inflorescence fragments of Z. nimmonii respectively and identified by the DNA sequence of the ITS regions. Both strains were subjected to fermentation in liquid media and extracted with ethyl acetate (EA) solvent, and evaporated to dryness in Rotary Flash Evaporator and designated as crude dry extracts. The extracts (250 µg/ml) were dissolved in EA and tested for antibacterial potential against two Gram-positive and four Gram-negative test bacterial strains seeded onto Mueller–Hinton agar (MH) by the disc diffusion assay. The plates were incubated for 24 h at 35 ±  °C and the presence of or absence of the inhibitory zone around the disk was measured and photographed. S—Streptomycin disk, C—Negative control

Thin layer chromatography (TLC) and bio-autography

The extract of B. specifera was characterized for the presence of antibacterial compounds by TLC and bio-autography methods. This analysis aided direct visualisation of the presence of active compounds as inhibition zone at Rf-0.56 on the chromatogram, which was collected carefully and characterized by liquid chromatography (LC), gas chromatography (GC) and mass spectral analysis.

Identification of antibacterial compounds through LC–MS and GC–MS analysis

The identification of phenolic compound was done by LC–MS while the GC–MS was carried out to identify volatile and aliphatic compounds. LC–MS data revealed the presence of caffeic acid in the TLC fraction of the extracts. GC–MS of the TLC fraction revealed six peaks corresponding to the presence of six compounds viz., (1) Bicyclo[3.2.0]heptan-2-one, 6-hydroxy-5-methyl-6-vinyl; (2) Adipic acid divinyl ester; (3) 1,4-Naphthoquinone, 6-acetyl-2,5-dihydroxy; (4) decanedioic acid, 3,7-dimethyl ester; (5) (Z)-4-Hexenoic acid 2-acetyl-2-methyl-ethyl ester and (6) Butanoic acid 2-acetyl-3-methyl-methyl ester (Table 3). The chromatograms of liquid and gas chromatography with mass spectra is represented in Fig. 2. Caffeic acid (3,4-dihydroxycinnamic acid) is a simple phenolic acid derived from hydroxycinnamic acid. This compound is attributed in biological properties viz., antibacterial, fungicidal and antioxidative (Gulcin 2006; Bozic et al. 2012; Pinho et al. 2015), though the antioxidant capacity has been studied the most. However, Pinho et al. (2015) reported the antibacterial capacity of encapsulated caffeic acid against K. pneumoniae and S. aureus.

Table 3.

Identification of antibacterial compounds from B. specifera extract by GC–MS

Peak Retention time (min) Molecular weight Chemical structure Name of the compound % Area
1 25.02 166.22 C10H14O2 Bicyclo[3.2.0]heptan-2-one, 6-hydroxy-5-methyl-6-vinyl- 36.9
2 26.94 198.26 C11H18O3 (Z)-4-Hexenoic acid, 2-acetyl-2-methyl-, ethyl ester 3.5
3 27.21 198.21 C10H14O4 Adipic acid divinyl ester 15.1
4 28.69 232.19 C12H8O5 1,4-Naphthoquinone, 6-acetyl-2,5-dihydroxy 9.5
5 30.27 230.30 C12H22O4 Decanedioic acid, 3,7-dimethyl-, dimethyl ester 5.1
6 31.00 158.19 C8H14O Butanoic acid, 2-acetyl-3-methyl-, methyl ester 2.9
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Fig. 2.

Fig. 2

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Chromatograms of antibacterial compounds from the TLC positive fraction of B. specifera. B. specifera was isolated as an endophyte of Z. nimmonii root fragment and cultured in PD broth to obtain the secondary metabolites. The culture filtrate was extracted with ethyl-acetate (EA3X) and concentrated by flash evaporation to obtain EA crude dry extract, which was subsequently tested positive for the antibacterial activity against a panel of test bacterial strains. Therefore, the crude EA extract was subjected to TLC for the separation of active compounds in a mixture of chloroform: ethyl acetate: formic acid (20:16:4) and confirmed for the zone of inhibition with the agar overlay bioautography method. The TLC-positive fraction from the preparative TLC plate was analyzed for the presence of volatile and aliphatic compounds by GC–MS. The compounds were identified from the chromatogram by computer matching of their mass spectral fragmentation patterns with the NIST-MS library. LC–electrospray ionization (ESI)–MS were carried out using mass spectroscopy (Waters, Milford, MA, USA) for the detection of phenolic compound. LC separation was performed on a reversed-phase Acquity UPLC BEH C18 column. A GC of volatile compounds, (1) Bicyclo[3.2.]heptan-2-one, 6-hydroxy-5-methyl-6-vinyl-, (2) Z-4-Hexenoic acid, 2-acetyl-2-methyl, ethyl ester, (3) Adipic acid divinyl ester, (4)1,4-Naphthoquinone, 6-acetyl-2,5-dihydroxy, (5) Decanedioic acid, 3,7-dimethyl, dimethyl ester, (6) Butanoic acid, 2-acetyl-3-methyl, methyl ester; B LC of phenolic compound, 7- Caffeic acid.

GC–MS analysis showed the presence of Bicyclo [3.2.0] heptan-2-one-6-hydroxy-5-methyl-6-vinyl (36.9%); Adipic acid ester (15.1%); 1,4-Naphthoquinone, 6-acetyl-2,5-dihydroxy (9.5%) and Decanedioic acid-3,7-dimethyl ester (5.1%) as major compounds. In corroboration with our observation, Abdelshafeek et al. (2010) reported the antibacterial activity of Verbena tenuisecta volatile oil and found Bicyclo [3.2.0] heptan-2-one-6-hydroxy-5-methyl-6-vinyl as one of the major compounds in GC analysis. Adipic acid ester is a dicarboxylic acid ester. Jae-Kweon et al. (1997) showed that Adipic acid in combination with monoglycerides, had activity against Gram positive and Gram negative strains. The occurrence of adipic acid is rare in nature (Musser, 2005), and the presence of this compound in the endophytic extract is worthy.

1,4-Naphthoquinone-6-acetyl-2,5-dihydroxy is a derivative of Naphthoquinone, structurally related to naphthalene. Naphthoquinone derivatives exhibit important biological activities such as antibacterial, antifungal, anti-parasitic and antiviral (Ramos-Peralta et al. 2015). Different antibiotics viz., rifamycin, tolypomycin, damavaricin and manumycin possess the quinone ring as pharmacophore. The inhibition of Gram positive and Gram negative bacteria by 1,4-Naphthoquinone is attributed to its pharmacophore (Ramos-Peralta et al. 2015). The antibacterial activity of 1,4-Naphthoquinone and its derivatives against Gram positive bacteria is documented (Brandelli et al. 2004; Lim et al. 2007). Endophytic strains viz., A. terreus and N. haematococca also displayed significant inhibition against pathogens except B. subtilis. The phenolic compounds, Chlorogenic acid and quercetin were detected in the extracts of A. terreus and N. haematococca (Das et al. 2017a) and possess antibacterial activity. Both these compounds are reported as excellent antibacterial agents (Hirai et al. 2010; Lou et al. 2011), which substantiates our findings.

To the best of our knowledge this is the first report on the antibacterial activity and identification of active compounds from the fungal endophyte of Z. nimmonii. B. specifera is a potential antibacterial endophyte, as the extract contained newer source of active compounds such as 1,4-Naphthoquinone- 6-acetyl-2,5-dihydroxy, Bicyclo[3.2.0]heptan-2-one, 6-hydroxy-5-methyl-6-vinyl and Adipic acid ester. Caffeic acid is a potential antimicrobial agent. Hence, this study envisages possible drug discovery using endophytes from the traditional medicinal plants.

Acknowledgements

The authors thank the Institution of Excellence-Biodiversity Project, University of Mysore, and the facilities utilized for the study.

Author contributions

MD contributed the experimental part on the isolation and characterization of endophytic fungi, testing for antibacterial potentials and characterization studies as well as the writing and the presentation of manuscript. HSP carried out the statistical design of the study and overall supervision, while the MSN is credited with the collection of the endemic plant Z. nimmonii, from Western Ghats and the identification of the specimen and the approval of the manuscript in the final form.

Funding

This work was supported by University Grants Commission–Major Research Project, Govt. of India with the Grant Number F. No. 40-307/2011 (SR) dt. 30-06-2011.

Compliance with ethical standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Ethical statements

No experimental animals were used in this study.

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